Antibodies to leukemia inhibitory factor

ABSTRACT

The invention relates to monoclonal antibodies to human leukemia inhibitory factor. The disclosed monoclonal antibodies are believed to recognize unique epitopes on hLIF and are useful in the treatment of conditions wherein the presence of hLIF causes or contributes to undesirable pathological effects, such as cachexia, dysregulated calcium metabolism, or excessive bone cell proliferation, and in the detection of hLIF, for example, in clinical samples or specimens.

This is a continuation of application Ser. No. 08/438,455 filed on May10, 1995, now U.S. Pat. No. 5,668,003 issued Sep. 16, 1997, which is adivisional of application Ser. No. 08/258,918 filed on Jun. 13, 1994,now U.S. Pat. No. 5,688,681 issued Nov. 18, 1997, which is acontinuation of application Ser. No. 08/056,966 filed Apr. 29, 1993, nowabandoned which is a continuation of Ser. No. 07/880,400, filed May 8,1992, now abandoned which applications are incorporated herein byreference and to which application(s) priority is claimed under 35 USC §120.

FIELD OF THE INVENTION

This application relates to hybrid cell lines (lymphocyte hybridomas)for the production of monoclonal antibodies to human leukemia inhibitoryfactor, to such homogeneous monospecific antibodies, and to the use ofsuch antibodies for diagnostic and therapeutic purposes.

BACKGROUND OF THE INVENTION

Leukemia inhibitory factor (LIF) is a polypeptide with a broad range ofbiological effects. LIF was initially purified from mouse cells andidentified on the basis of its ability to induce differentiation in andsuppress the proliferation of the murine monocytic leukemia cell lineM1. Tomida, et al., J. Biol. Chem. 259:10978-10982 (1984); Tomida, etal., FEBS Lett. 178:291-296 (1984). Human LIF (hLIF) subsequently wasshown to have comparable effects on human HL60 and U937 cells,particularly when acting in collaboration with GM-CSF or G-CSF colonystimulating factors. Maekawa, et al. Leukemia 3:270-276 (1989).

LIF has been shown to exhibit a variety of biological activities andeffects on different cell types. For example, it has been shown tostimulate osteoblast proliferation and new bone formation, Metcalf, etal., Proc. Nat. Acad. Sci., 86:5948-5952 (1989), as well as boneresorption, Abe, et al., Proc. Nat. Acad. Sci. 83:5958-5962 (1986);Reid, et al., Endocrinology 126:1416-1420 (1990), stimulate liver cellsto produce acute phase plasma proteins, Baumann, et al., J. Immunol.143:1163-1167 (1989), inhibit lipoprotein lipase, Mori, et al., Biochem.Biophys. Res. Commun. 160:1085-1092 (1989), stimulate neuronaldifferentiation and survival, Murphy, et al., Proc. Nat. Acad. Sci.88:3498-3501 (1991), Yamamori, et al., Science 246:1412-1416 (1989), andinhibit vascular endothelial cell growth, Ferrara, et al., Proc. Nat.Acad. Sci. 89:698-702 (1992). Receptors for LIF have been found onmonocyte-macrophages, osteoblasts, placental trophoblasts, and liverparenchymal cells. Hilton, et al., J. Cell. Biochem. 46:21-26 (1991);Allan, et al., J. Cell. Physiol. 145:110-119 (1990); Hilton, et al.,Proc. Nat. Acad. Sci. 85:5971-5975 (1988).

Depending upon its particular activity or effect, LIF has been referredto by various names, including differentiation-inducing factor (DIF,D-factor), hepatocyte-stimulating factor (HSF-II, HSF-III),melanoma-derived LPL inhibitor (MLPLI), and cholinergic neuronaldifferentiation factor (CDF). Hilton, et al., J. Cell. Biochem. 46:21-26(1991).

Genomic and cDNA clones encoding murine, rat, and human LIF have beenisolated. Gearing, et al., EMBO J. 6:3995-4002 (1987); Yamamori, et al.,Science 246:1412-1416 (1989); Gough, et al., Proc. Nat. Acad. Sci.85:2623-2627 (1988).

Antibodies to hLIF are expected to have valuable diagnostic andtherapeutic applications, such as in assaying for the presence of hLIFin clinical specimens, and in regulating the biological effects of hLIFand the interaction of hLIF with its receptors and other ligands. Inparticular, monoclonal antibodies (mAbs) detecting unique epitopes ofhLIF would be of great value in understanding and regulating the diversebiological activities of hLIF. Neutralizing mAbs specific for hLIF thatinhibit one or more of the biological activities or effects of hLIF havegreat potential as therapeutic agents useful in the treatment ofconditions wherein the presence of hLIF causes or contributes toundesirable pathological effects, such as cachexia, dysregulated calciummetabolism, or excessive bone resorption (such as may be associated withosteoporosis).

Several polyclonal antibodies have been described that react with LIF.Tomida, et al., FEBS Letters 151:281-285 (1983) immunized rabbits withpartially purified D-factor from mouse cells, and obtained antibodiescapable of neutralizing the activity of mouse D-factor, and to a lesserextent, rat and hamster D-factors, in several assays. Baumann, et al.,J. Immunol. 143:1163-1167 (1989) reported that rabbit polyclonalantibodies against hepatocyte-stimulating factor III (HSF-III)neutralized that activity of hLIF on hepatic cells.

There is a need for high affinity monoclonal antibodies to hLIF that arecapable of effective inhibition of the biological activities of hLIF. Itwould be particularly desirable to provide monoclonal antibodies thatare effective inhibitors of hLIF binding to its receptors, but which donot interfere with the binding of other factors, such as interleukin 1(IL-1), interleukin 3 (IL-3), interleukin 6 (IL-6), tumor necrosisfactor-α (TNF-α), granulocyte CSF (G-CSF), granulocyte-macrophage-colonystimulating factor (GM-CSF), and Oncostatin M.

SUMMARY OF THE INVENTION

The present invention is based on successful research involving theproduction and extensive characterization of monoclonal antibodies tohLIF. Accordingly, the present invention is directed to monoclonalantibodies, and derivatives thereof, which are capable of recognizingunique epitopes on hLIF and/or which exhibit high affinity for hLIF. Theinvention is further directed to monoclonal antibodies capable ofinhibiting one or more of the biological activities of hLIF.

In one aspect, the invention concerns an anti-hLIF monoclonal antibodythat is capable of inhibiting the mitogenic effect of hLIF on leukemiccells, and that does not cross-react with IL-1, IL-3, IL-6, TNF-α,G-CSF, or GM-CSF.

In another aspect, the invention concerns isolated nucleic acid encodingsuch antibodies, and hybridoma or recombinant cells producing suchantibodies.

In a further aspect, the invention concerns the therapeutic ordiagnostic use of such antibodies. The monoclonal antibodies of theinvention are useful as therapeutic agents, either by themselves or inconjunction with (chemo)therapeutic agents, to treat diseases orconditions that are aggravated by hLIF. The monoclonal antibodies of theinvention also are useful in diagnostic and analytical assays fordetermining the presence of hLIF in clinical specimens.

These and further aspects will be apparent from the following detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the binding (antigenic) specificity of anti-hLIF mAbD25.1.4 as determined by ELISA.

FIGS. 2A and 2B shows a comparison of the binding of anti-hLIFmonoclonal antibodies to recombinant human LIF (rHuLIF) and recombinantmurine LIF (rMuLIF) as determined by ELISA.

FIG. 3 shows the binding of horse radish peroxidase conjugated anti-hLIFmonoclonal antibodies (HRP-mAbs) to hLIF in the presence and absence of100-fold molar excess of unlabeled anti-hLIF monoclonal antibody(D3.14.1, D4.16.9, D25.1.4, or D62.3.2) or irrelevant control antibody(anti-hVEGF mAb A3.13.1).

FIG. 4 shows the effect of anti-hLIF monoclonal antibody (D3.14.1,D4.16.9, D25.1.4, or D62.3.2) or irrelevant control antibody (anti-hVEGFmAb A3.13.1) on hLIF inhibition of M1-T22 murine myeloid leukemic cellgrowth. Results are expressed as a percentage reduction (neutralization)of such hLIF activity as compared to a control assay in which hLIFinhibition of M1-T22 murine myeloid leukemic cell growth was determinedin the absence of antibody.

FIGS. 5A-5F shows fluorescence activated cell sorting (FACS) analysis ofintracellular Ca²⁺ levels in Jurkat human T-cells exposed to hLIF alone(no Ab), or hLIF pre-incubated with anti-hLIF monoclonal antibody(D3.14.1, D4.16.9, D25.1.4, or D62.3.2) or irrelevant control antibody(anti-hVEGF mAb A3.13.1). The assay was carried out over three minutestime; hLIF alone or the pre-incubated mixtures of hLIF and antibody wereadded one minute after the start of the FACS analysis (in the firstpanel (no Ab), for example, the time of addition of hLIF is indicated bythe downward pointing arrow).

FIG. 6 shows the binding of ¹²⁵ I-hLIF to hLIF receptors on M1-T22 cellsin the presence of unlabeled anti-hLIF monoclonal antibody (D3.14.1,D4.16.9, D25.1.4, or D62.3.2) or irrelevant control antibody (anti-hVEGFmAb 3.13.1). The amount of anti-hLIF monoclonal antibody added waseither 50×, 10×, or 1× molar amount of hLIF in the assay. NSC=¹²⁵ I-hLIFbound to M1-T22 cells in the presence of 1000-fold molar excess ofunlabeled hLIF (a measure of non-specific binding). TC=¹²⁵ I-hLIF boundto M1-T22 cells in the absence of antibody (a measure of maximalbinding).

FIG. 7 shows the results of an ELISA for detection of variousconcentrations of hLIF, using mAb 4.16.9 as a capture antibody and mAb3.14.1 as a detection antibody.

DETAILED DESCRIPTION OF THE INVENTION A. Definitions and General Methods

The term "monoclonal antibody" as used herein refers to a substantiallyhomogeneous population of antibodies, i.e., the individual antibodiescomprising the population are identical in specificity and affinityexcept for possible naturally occurring mutations that may be present inminor amounts. Note that a monoclonal antibody composition may containmore than one monoclonal antibody.

The monoclonal antibodies included within the scope of the inventioninclude hybrid and recombinant antibodies (for example, "humanized"antibodies) regardless of species of origin or immunoglobulin class orsubclass designation, as well as antibody fragments (for example, Fab,F(ab')₂, and Fv), so long as they have the novel and unobviouscharacteristics of the antibodies described herein, in preferredembodiments being antibodies that are capable of binding tosubstantially the same epitope as one recognized by a monoclonalantibody produced by any one of the D3.14.1, D4.16.9, D25.1.4, orD62.3.2 hybridomas described herein, and/or that have affinity for thatepitope which is greater than or equal to the affinity of a monoclonalantibody produced by any one of such hybridomas.

Thus, the modifier "monoclonal" indicates the character of the antibodyas a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies of the invention may bemade using the hybridoma method first described by Kohler & Milstein,Nature 256:495-497 (1975), or may be made by recombinant DNA methods.For example, see Cabilly, et al., U.S. Pat. No. 4,816,567; or Mage &Lamoyi, in Monoclonal Antibody Production Techniques and Applications,pp. 79-97 (Marcel Dekker, Inc., New York, 1987).

In the hybridoma method, a mouse or other appropriate host animal isimmunized with hLIF by subcutaneous, intraperitoneal, or intramuscularroutes to elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the protein used forimmunization. Alternatively, lymphocytes may be immunized in vitro.Lymphocytes then are fused with myeloma cells using a suitable fusingagent, such as polyethylene glycol, to form a hybridoma cell. Goding,Monoclonal Antibodies: Principles and Practice, pp.59-103 (AcademicPress, 1986).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh level expression of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such asP3-NS-1-Ag-4-1, Kohler, et al., Eur. J. Immunol 6:292-295 (1976),X63-Ag8.653, Kearney, et al., J. Immunol. 123:1548-1550 (1979),SP2/0-Ag/4, Sltulman, et al., Nature 276:269-270 (1978), or P₃ X63Ag8U₁Yelton et al., Curr. Top. Microbiol. Immunol. 81:1-7 (1978). Humanmyeloma and mouse-human heteromyeloma cell lines also have beendescribed for the production of human monoclonal antibodies. Kozbor, J.Immunol. 133:3001-3005 (1984). Brodeur, et al., Monoclonal AntibodyProduction Techniques and Applications, pp.51-63 (Marcel Dekker, Inc.,New York, 1987).

Culture medium in which hybridoma cells are growing is convenientlyassayed for production of monoclonal antibodies directed against hLIF.Preferably, the binding specificity of antibodies is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA),or by fluorescence activated cell sorting (FACS). The monoclonalantibodies of the invention are those that preferentially bind tosoluble or cell bound hLIF and which are neutralizing, as explainedherein. The specificity of binding of the monoclonal antibodies of theinvention is determined by reaction of the antibodies with factors otherthan hLIF, with the objective being the identification of antibodiesthat do not bind to any factors other than hLIF, especially IL-1, IL-3,IL-6, G-CSF, GM-CSF, or Oncostatin M. A monoclonal antibody thatpreferentially binds to soluble or cell bound hLIF generally willexhibit at least the same degree of specificity of binding as amonoclonal antibody produced by any one of the D3.14.1, D4.16.9,D25.1.4, or D62.3.2 hybridomas described herein.

In a preferred embodiment of the invention, the monoclonal antibody willhave an affinity which is greater than about 10⁹ liters/mole andpreferably is equal to or greater than about 10¹⁰ liters/mole, asdetermined, for example, by the Scatchard analysis of Munson, et al.,Anal. Biochem. 107:220-239 (1980).

The term "neutralizing antibody" as used herein refers to a monoclonalantibody that is capable of substantially inhibiting or eliminating abiological activity of hLIF.

After hybridoma cells are identified that produce neutralizingantibodies of the desired specificity and affinity, the clones typicallyare subcloned by limiting dilution procedures and grown by standardmethods. Goding, Monoclonal Antibodies: Principles and Practice,pp.59-104 (Academic Press, 1986). Suitable culture media for thispurpose include, for example, Dulbecco's Modified Eagle's Medium orRPMI-1640 medium. In addition, the hybridoma cells may be grown in vivoas ascites tumors in an animal.

The monoclonal antibodies secreted by the selected hybridoma cells aresuitably purified from cell culture medium or ascites fluid byconventional immunoglobulin purification procedures such as, forexample, protein A-SEPHAROSE™, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies of the invention is readilyisolated and sequenced using conventional procedures (for example, byusing oligonucleotide probes that are capable of binding specifically togenes encoding the heavy and light chains of murine antibodies). Thehybridoma cells of the invention serve as a preferred source of suchDNA. Once isolated, the DNA is ligated into expression or cloningvectors, which are then transfected into host cells such as simian COScells, Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein. The transformant cells arecultured to obtain the synthesis of monoclonal antibodies in therecombinant host cell culture.

The DNA optionally is modified in order to change the character of theimmunoglobulin produced by its expression. Immunoglobulin variants arewell known. For example, chimeric antibodies are made by substitutingthe coding sequence for human heavy and light chain constant domains inplace of the homologous murine sequences. Cabilly, et al., U.S. Pat. No.4,816,567 et al.; Morrison, et al., Proc. Nat. Acad. Sci. 81:6851-6855(1984). In addition, the Fc domain chosen is any of IgA, IgG-1, -2, -3or -4. The Fc domain optionally is capable of effector functions such ascomplement binding.

Humanized forms of the murine antibodies are made by substituting thecomplementarity determining regions of the mouse antibody into a humanframework domain, as described, for example, in U.S. patent applicationSer. No. 07/715,272, now abandoned. In some embodiments, selected murineframework residues also are substituted into the human recipientimmunoglobulin.

Fusions of the antibodies of this invention and cytotoxic moieties aremade, for example, by ligating to the antibody coding sequence all orpart of the coding sequence for a cytotoxic non-immunoglobulinpolypeptide. Such non-immunoglobulin polypeptides include polypeptidetoxins such as ricin, diphtheria toxin, or Pseudomonas exotoxin. Also,the conjugates can be prepared by in vitro methods. For example,immunotoxins may be constructed using a disulfide exchange reaction orby forming a thioether bond between the antibody and the toxinpolypeptide. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate. Suitable fusionpartners for the antibodies of this invention include viral sequences,cellular receptors such as the T-cell receptor, cytokines such as TNF,interferons, or interleukins, and other biologically or immunologicallyactive polypeptides. Typically such non-immunoglobulin fusionpolypeptides are substituted for the constant domains of an antibody ofthe invention. Alternatively, they are substituted for the variabledomains of one antigen-combining site of an antibody of the invention.

Substitution of the Fc or complementary determining regions (CDRs) of anantibody having specificity for non-hLIF antigen will create a chimericbivalent antibody comprising one antigen-combining site havingspecificity for hLIF and another antigen-combining site havingspecificity for a different antigen. In such embodiments, the lightchain is deleted and the Fv domain of the heavy chain is substitutedwith the desired polypeptide. These antibodies are termed bivalent orpolyvalent, depending upon the number of immunoglobulin "arms" possessedby the Fc domain employed (IgMs will be polyvalent). An antibody alsomay be rendered multivalent by intracellular recombination of antibodieshaving more than one specificity. For instance, an antibody in someembodiments is capable of binding hLIF as described elsewhere herein butis also capable of binding a T-cell, osteoblast, or liver cell surfaceantigen. In the case of T-cells, such antigens include CD3, CD4, CD8,CD18, CD11a, CD11b or CD11c. Examples of antibodies to cell surfaceantigens are well known. The multispecific, multivalent antibodies aremade by cotransforming a cell with DNA encoding the heavy and lightchains of both the anti-hLIF antibody and the anti-cell surface antigenantibody. Those expressed antibodies having the desired multispecific,multivalent structure then are recovered by immunoaffinitychromatography or the like. Alternatively, such antibodies are made frommonovalent antibodies which are recombined in vitro in conventionalfashion.

Monovalent antibodies also are made by techniques that are conventionalper se. Recombinant expression of light chain and a modified heavy chainis suitable. The heavy chain is truncated generally at any point in theFc region so as to prevent heavy chain crosslinking. Alternatively, therelevant cysteines are substituted with another residue or deleted so asto prevent crosslinking. In vitro methods also are used to producemonovalent antibodies. For example, Fab fragments are prepared byenzymatic cleavage of intact antibody.

For diagnostic applications, the antibodies of the invention typicallywill be labeled with a detectable moiety. The detectable moiety can beany one which is capable of producing, either directly or indirectly, adetectable signal. For example, the detectable moiety may be aradioisotope, such as ³ H, ¹⁴ C, ³² P, ³⁵ S, or ¹²⁵ I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; radioactive isotopic labels, such as, forexample, ¹²⁵ I, ³² P, ¹⁴ C, technicium, or ³ H; or an enzyme, such asalkaline phosphatase, beta-galactosidase or horseradish peroxidase.

Any method known in the art for separately conjugating the antibody tothe detectable moiety may be employed, including those methods describedby Hunter, et al., Nature 144:945 (1962); David, et al., Biochemistry13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219 (1981); andNygren, J. Histochem. and Cytochem. 30:407 (1982).

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc., 1987).

Competitive binding assays rely on the ability of a labeled standard(which may be hLIF or an immunologically reactive portion thereof) tocompete with the test sample analyte (hLIF) for binding with a limitedamount of antibody. The amount of hLIF in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insoluble threepart complex. David & Greene, U.S. Pat. No. 4,376,110. The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme.

The antibodies of the invention also are useful for in vivo imaging,wherein an antibody labeled with a detectable moiety such as aradio-opaque agent or radioisotope is administered to a host, preferablyinto the bloodstream, and the presence and location of the labeledantibody in the host is assayed. This imaging technique is useful in thestaging and treatment of neoplasms or bone disorders. The antibody maybe labeled with any moiety that is detectable in a host, whether bynuclear magnetic resonance, radiology, or other detection means known inthe art.

For therapeutic applications, the antibodies of the invention areadministered to a mammal, preferably a human, in a pharmaceuticallyacceptable dosage form. They are administered intravenously as a bolusor by continuous infusion over a period of time, by intramuscular,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. When the antibody possesses the suitableactivity it is also suitably administered by intratumoral, peritumoral,intralesional, or perilesional routes, to exert local as well assystemic therapeutic effects.

Such dosage forms encompass pharmaceutically acceptable carriers thatare inherently nontoxic and nontherapeutic. Examples of such carriersinclude ion exchangers, alumina, aluminum stearate, lecithin, serumproteins, such as human serum albumin, buffers such as phosphate orglycine, sorbic acid, potassium sorbate, partial glyceride mixtures ofsaturated vegetable fatty acids, water, salts, or electrolytes such asprotamine sulfate, sodium chloride, metal salts, colloidal silica,magnesium trisilicate, polyvinyl pyrrolidone, cellulosic polymers, andpolyethylene glycol. Carriers for topical or gel-based forms of antibodyinclude polysaccharides such as sodium carboxymethylcellulose ormethylcellulose, polyvinylpyrrolidone, polyacrylates,polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol,and wood wax alcohols. Conventional depot forms include, for example,microcapsules, nano-capsules, liposomes, plasters, sublingual tablets,and polymer matrices such as polylactide:polyglycolide copolymers. Whenpresent in an aqueous dosage form, rather than being lyophilized, theantibody typically will be formulated at a concentration of about 0.1mg/ml to 100 mg/ml, although wide variation outside of these ranges ispermitted.

For the prevention or treatment of disease, the appropriate dosage ofantibody will depend on the type of disease to be treated, as definedabove, the severity and course of the disease, whether the antibodiesare administered for preventive or therapeutic purposes, the course ofprevious therapy, the patient's clinical history and response to theantibody, and the discretion of the attending physician. The antibody issuitably administered to the patient at one time or over a series oftreatments.

Depending on the type and severity of the disease, about 0.015 to 15 mgof antibody/kg of patient weight is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. For repeatedadministrations over several days or longer, depending on the condition,the treatment is repeated until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful and arenot excluded herefrom.

According to another embodiments of the invention, the effectiveness ofthe antibody in preventing or treating disease may be improved byadministering the antibody serially or in combination with another agentthat is effective for the same clinical objective, such as anotherantibody directed against a different epitope than the principalantibody, or one or more conventional therapeutic agents known for theintended therapeutic indication, e.g. prevention or treatment ofconditions associated with excessive bone resorption such asosteoporosis.

The antibodies of the invention also are useful as affinity purificationagents. In this process, the antibodies against hLIF are immobilized ona suitable support, such as SEPHADEX™ resin or filter paper, usingmethods well known in the art. The immobilized antibody then iscontacted with a sample containing the hLIF to be purified, andthereafter the support is washed with a suitable solvent that willremove substantially all the material in the sample except the hLIF,which is bound to the immobilized antibody. Finally, the support iswashed with another suitable solvent, such as glycine buffer, pH 5.0,that will release the hLIF from the antibody.

The following examples are offered by way of illustration only and arenot intended to limit the invention in any manner. All patent andliterature references cited throughout the specification are expresslyincorporated.

EXAMPLE 1 Preparation of Monoclonal Antibodies

Recombinant hLIF, Schmelzer, et al., Prot. Exp. Purificat. 1:54-62(1990), was conjugated to keyhole limpet hemocyanin (KLH) according tothe method of Nicolson, et al. Proc. Nat. Acad. Sci. 68:942 (1971).Balb/c mice were injected intraperitoneally with 10 μg of the resultingKLH-hLIF conjugate three times at two week intervals, and were boostedwith the same dose of KLH-hLIF conjugate four days prior to cell fusion.

Spleen cells from the immunized mice were fused with P₃ X63Ag8U₁ myelomacells, Yelton, et al., Curr. Top. Microbiol. Immunol. 81:1-7 (1978),using 35% polyethylene glycol (PEG) as described. Laskov, et al., Cell.Immunol. 55:251-264 (1980). Hybridomas were selected in HAT medium.

Supernatants from hybridoma cell cultures were screened for anti-hLIFantibody production by an ELISA assay using hLIF-coated microtiterplates. Antibody that was bound to hLIF in each of the wells wasdetermined using alkaline phosphatase-conjugated goat anti-mouse IgGimmunoglobulin and the chromogenic substrate p-nitrophenyl phosphate.Harlow & Lane, Antibodies: A Laboratory Manual, p.597 (Cold SpringHarbor Laboratory, 1988). Hybridomas thus determined to produceanti-hLIF monoclonal antibodies were subcloned by limiting dilution.

Initially, 65 hybridomas producing anti-hLIF monoclonal antibodies wereidentified by ELISA. Of those, four hybridomas, designated D3.14.1,D4.16.9, D25.1.4, and D62.3.2 were chosen for further characterization.Ascites were produced in Balb/c mice and monoclonal antibodies werepurified using protein-G conjugated 4B SEPHAROSE™. Hereinafter, themonoclonal antibodies produced by the D3.14.1, D4.16.9, D25.1.4, andD62.3.2 hybridomas (designated ATCC accession no. HB 11076, ATCCaccession no. HB 11077, ATCC accession no. HB 11074, and ATCC accessionno. HB 11075, respectively, and deposited on Jun. 23, 1992, with theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va. 20110-2209) are referred to as mAb D3.14.1, mAb D4.16.9, mAbD25.1.4, and mAb D62.3.2 respectively.

EXAMPLE 2 Characterization of Monoclonal Antibodies A. Antigen BindingSpecificity

The binding specificities of the anti-hLIF monoclonal antibodiesproduced by the D3.14.1, D4.16.9, D25.1.4, and D62.3.2 hybridomas weredetermined by ELISA. 10 μg/ml of purified monoclonal antibody was addedto the wells of microtiter plates that previously has been coated with 2μg of hLIF, human IL-1, human IL-3, human IL-6, human TNF-α, humanG-CSF, human GM-CSF, or human Oncostatin M. IL-6, G-CSF, and OncostatinM are known to share significant amino acid sequence homologies withhLIF. Rose, et al., Proc. Nat. Acad. Sci. 88:8641-8645 (1991).

Bound antibody was detected with horse radish peroxidase (HRP)conjugated goat anti-mouse IgG immunoglobulins. The HRP color reactionwas developed by the addition of phosphate buffered saline (PBS)containing 0.4 mg/ml. of o-phenylenediamine diamine dihydrochloride plus0.4 μl/ml 30% hydrogen peroxide. The reaction was stopped by theaddition of 100 μl/well 2.25 M sulfuric acid. The color reaction wasmeasured at 490 nm with an ELISA plate reader.

The results of those assays showed that each of mAb D3.14.1, mAbD4.16.9, mAb D25.1.4, and mAb D62.3.2 binds to hLIF, and not appreciablyto those other protein factors, except that mAb D3.14.1, showed a weakcross-reactivity with human Oncostatin M. The results of the assay ofmAb D25.1.4 reactivity with various protein factors are shown in FIG. 1.The other monoclonal antibodies showed similar binding specificity forhLIF.

Additionally, the binding of mAb D3.14.1, mAb D4.16.9, mAb D25.1.4, andmAb D62.3.2 to murine LIF was determined by ELISA. As shown in FIGS. 2Aand 2B, mAb D4.16.9 and mAb D62.3.2 bound to murine LIF with about thesame affinity as to hLIF, mAb D25.1.4 bound to murine LIF with loweraffinity than to hLIF, and mAb D3.14.1 did not detectably bind to murineLIF.

B. Epitope Mapping

A competitive binding ELISA was used to determine whether the monoclonalantibodies produced by the D3.14.1, D4.16.9, D25.1.4, and D62.3.2hybridomas bind to the same or different epitopes (sites) within hLIF.Anti-hLIF antibodies (mAb D3.14.1, mAb D4.16.9, mAb D25.1.4, and mAbD62.3.2) and an irrelevant anti-human vascular endothelial growth factor(hVEGF) antibody (mAb A3.13.1) were conjugated with horse radishperoxidase (HRP) and the competitive binding ELISA was performed asdescribed by Kim, et al., Infect. Immun. 57:944-950 (1989).

As shown in FIG. 3, the inhibition pattern of the binding of eachHRP-conjugated anti-hLIF antibody was unique, making it likely that eachantibody recognizes a different epitope on hLIF. The binding of each ofmAb D3.14.1 and mAb D25.1.4 was inhibited only by itself. The binding ofmAb D4.16.4 was inhibited by mAb D3.14.1 and mAb D25.1.4 as well as mAbD4.16.4, while the binding of mAb D3.14.1 and mAb D25.1.4 was notblocked by mAb D4.16.9. The binding of mAb D62.3.2 was inhibited by mAbD25.1.4, but the binding of mAb D25.1.4 was not inhibited by mAbD62.3.2.

C. Isotyping

The isotypes of the anti-hLIF monoclonal antibodies produced by theD3.14.1, D4.16.9, D25.1.4, and D62.3.2 hybridomas were determined byELISA. Hybridoma cell culture supernatants were added to the wells ofmicrotiter plates that had previously been coated with hLIF. Thecaptured anti-hLIF monoclonal antibodies were incubated with differentisotype-specific alkaline phosphatase-conjugated goat anti-mouseimmunoglobulins (Fisher Biotech, Pittsburgh, Pa., USA), and the bindingof the conjugated antibodies to the anti-hLIF monoclonal antibodies wasdetermined by the addition of p-nitrophenyl phosphate. The colorreaction was measured at 405 nm with an ELISA plate reader.

By that method, the isotype of the monoclonal antibodies produced byeach of the D3.14.1, D4.16.9, D25.1.4, and D62.3.2 hybridomas wasdetermined to be IgG1.

D. Binding Affinity

The affinities of the anti-hLIF monoclonal antibodies produced by theD3.14.1, D4.16.9, D25.1.4, and D62.3.2 hybridomas were determined by alcompetitive binding assays. A predetermined sub-optimal concentration ofmonoclonal antibody was added to samples containing 20,000-40,000 cpm¹²⁵ I-hLIF (1-2 ng) and various known amounts of unlabeled hLIF (1-1000ng) in 0.2 ml. phosphate buffered saline (PBS) containing 0.5% bovineserum albumin (BSA) and 0.05% Tween 20. After 1 hour at roomtemperature, 100 μl of goat anti-mouse Ig antisera (Pel-Freez, Rogers,Ark. USA) were added, and the mixtures were incubated another hour atroom temperature. Complexes of antibody and bound protein (immunecomplexes) were precipitated by the addition of 500 μl of 6%polyethylene glycol (PEG, mol. wt. 8000) at 4° C., followed bycentrifugation at 2000×G. for 20 min. at 4° C. The amount of ¹²⁵ I-hLIFbound to the anti-hLIF monoclonal antibody in each sample was determinedby counting the pelleted material in a gamma counter.

Affinity constants were calculated from the data by Scatchard analysis.Munson, et al., Anal. Biochem. 107:220 (1980). The affinity of each ofmAb D3.14.1, mAb D4.16.9, mAb D25.1.4, and mAb D62.3.2 was determined tobe in the range of about 1.4×10⁹ liters/mole to 1.7×10¹⁰ liters/mole.

F. Inhibition of hLIF Activity

The antibodies produced by the D3.14.1, D4.16.9, D25.1.4, and D62.3.2were assayed for their ability to neutralize the ability of hLIF toinhibit growth of M1-T22 murine myeloid leukemic cells, Tomida, et al.,Biochem. J. 176:655-669 (1978), and the ability of hLIF to inducerelease of intracellular calcium (Ca²⁺) from Jurkat human T-cells.M1-T22 is a subclone of the murine myeloid leukemia cell line M1,Tomida, et al., Biochem. J. 176:655-669 (1978). Jurkat human T-cellswere originally described by Weiss, et al., J. Immunol. 133:123-128(1984). The Jurkat human T-cells used in the assays described hereinwere from a stock maintained at Genetech, Inc.

The M1-T22 cell growth inhibition assay was carried out as described byLowe, et al., DNA 8:351-359 (1989). Generally, M1-T22 cells at 10⁴cells/were suspended in the wells of microtiter plates in minimalessential medium (MEM) supplemented with 10% fetal calf serum (FCS), 2mM nonessential amino acids, 1 mM sodium pyruvate, 1 mM glutamine,penicillin, and streptomycin, in the presence of 0.1-0.2 ng/mlrecombinant hLIF, with or without added anti-hLIF monoclonal antibodies(at 5×, 25×, or 125× molar excess relative to hLIF).

As shown in FIG. 4, mAb D25.1.4 blocked up to 87% of the M1-T22 growthinhibition activity of hLIF at a 1:5 molar ratio of antigen to antibody.mAb D25.1.4 blocked up to 90% of the M1-T22 growth inhibition activityof hLIF at a 1:25 molar ratio of antigen to antibody. mAb D3.14.1 andmAb D4.16.9 only minimally blocked the M1-T22 growth inhibition activityof hLIF.

In the course of making the present invention, it was found that hLIFinduces an increase in cytoplasmic calcium (Ca²⁺) concentration inJurkat human T-cells. To determine the ability of anti-hLIF monoclonalantibodies to inhibit that activity of hLIF, Jurkat human T-cells at 10⁶cells/ml were loaded with 5 μM indo-1 acetoxymethylester, Grynkiewicz,et al., J. Biol. Chem. 260:3440-3450 (1985), for 15 minutes at 37° C. asdescribed. June, et al., Pathol. Immunopatho. Res. 7:409-432 (1988). Thecells were then washed with RPMI medium without Ca²⁺ and Mg²⁺, andresuspended in the same medium. 10 μl of anti-hLIF monoclonal antibodywas incubated with 100 μl of hLIF (5 μg/ml) for one hour. hLIF alone oran antibody-hLIF mixture was then added to the indo-1 loaded Jurkat cellcultures. Immediately after, level of free cytosolic Ca²⁺ in the cellswas determined by fluorescence activated cell sorting, using a Coulter753 cell sorter and 200 mW UV excitation (351.1-363.8 nm), withfluorescence emission collected as a ratio of 405 nm/525 nm as describedby June, et al., Pathol. Immunopathol. Res. 7:409-432 (1988).

As shown in FIGS. 5A-5F, hLIF (not pre-incubated with antibody) inducedan increase in intracellular Ca²⁺ in the Jurkat cells within severalminutes after its addition to the cell cultures. Preincubation of hLIFwith any one of mAb D4.16.9, mAb D25.1.4, or mAb D62.3.3 reduced oreliminated the hLIF-induced increase in intracellular Ca²⁺.Preincubation of hLIF with mAb D3.14.1 had little or no effect onhLIF-induced increase in intracellular Ca²⁺.

To determine the effect of the monoclonal antibodies on hLIF binding toits receptors, ¹²⁵ I-hLIF binding to M1-T22 cells was compared in thepresence and absence of anti-hLIF monoclonal antibody. (5×10⁴ cpm/2 ng)was incubated with various amounts of antibody (either 1×, 10×, or 50×the molar amount of hLIF) in a final volume of 100 μl for 30 minutes at37° C. 10⁶ M1-T22 cells were then added to the antibody-hLIF mixture andincubation continued for 30 minutes at 37° C. Unbound ¹²⁵ I-hLIF wasseparated from bound ¹²⁵ I-hLIF by loading the mixtures onto a solutionof 20% sucrose, 0.1% bovine serum albumin (BSA) in phosphate bufferedsaline (PBS) and centrifuging at 300×G for 10 minutes. The supernatantwas removed and the radioactivity associated with the pellet was countedin a gamma counter.

As shown in FIG. 6, the anti-hLIF antibodies differed in their abilityto block hLIF binding to its receptors. At a 50× molar excess ofantibody to ¹²⁵ I-hLIF, mAb D25.1.4, mAb D62.3.2, mAb 3.14.1, and mAb4.16.9 reduced ¹²⁵ I-hLIF binding by about 95%, 65% 10%, and 30%,respectively. By comparison, ¹²⁵ I-hLIF binding to M1-T22 cells wasreduced about 80% by the addition of a 1000-fold molar excess ofunlabeled hLIF.

EXAMPLE 3 Use of Anti-HLIF Monoclonal Antibodies in ELISA to DetectHuman LIF

To determine levels of hLIF in clinical or other samples, and todistinguish hLIF from other protein factors, an ELISA was developedusing mAb 4.16.9 as a capture antibody and horse radishperoxidase-conjugated mAb 3.14.1 as a detection antibody. As shown inFIG. 7, using that combination of antibodies in an ELISA, as little as0.1 ng hLIF could be detected.

What is claimed is:
 1. A composition comprising:a monoclonal antibodythat specifically binds human Leukemia Inhibitory Factor and neutralizesa biological activity of human Leukemia Inhibitory Factor; and apharmaceutically acceptable carrier.
 2. A composition according to claim1, wherein the monoclonal antibody is competitively inhibited in itsbinding to human Leukemia Inhibitory Factor by a monoclonal antibodyselected from the group consisting of monoclonal antibodies produced byhybridoma cells deposited under American Type Culture CollectionAccession Numbers ATCC HB 11077, HB 11074, and HB
 11075. 3. Acomposition according to claim 1, wherein the monoclonal antibody iscapable of reducing the ability of human Leukemia Inhibitory Factor toinhibit growth of M1-T22 murine myeloid leukemic cells, wherein themonoclonal antibody is competitively inhibited in its binding to humanLeukemia Inhibitory Factor by a monoclonal antibody selected from thegroup consisting of monoclonal antibodies produced by hybridoma cellsdeposited under American Type Culture Collection Numbers ATCC HB 11074and HB
 11075. 4. A composition according to claim 1, wherein themonoclonal antibody is capable of reducing the ability of human LeukemiaInhibitory Factor to induce release of intracellular calcium from Jurkathuman T-cells, wherein the monoclonal antibody is competitivelyinhibited in its binding to human Leukemia Inhibitory Factor by amonoclonal antibody selected from the group consisting of monoclonalantibodies produced by hybridoma cells deposited under American TypeCulture Collection Accession Numbers ATCC HB 11074, ATCC HB 11075, andATCC HB
 11077. 5. A composition according to claim 1, wherein themonoclonal antibody has an affinity for human Leukemia Inhibitory Factorof at least 10⁹ liters/mole.
 6. A composition according to claim 1,wherein the monoclonal antibody is incapable of binding to Oncostatin M.7. A composition according to claim 1, wherein the monoclonal antibodyis produced by the hybridoma cell deposited under American Type CultureCollection Accession Number ATCC HB 11076 (referred to herein as3.14.1).
 8. A composition according to claim 1, wherein the monoclonalantibody is produced by the hybridoma cell deposited under American TypeCulture Collection Accession Number ATCC HB 11077 (referred to herein asD4.16.9).
 9. A composition according to claim 1, wherein the monoclonalantibody is produced by the hybridoma cell deposited under American TypeCulture Collection Accession Number ATCC HB 11074 (referred to herein asD25.1.4).
 10. A composition according to claim 1, wherein the monoclonalantibody is produced by the hybridoma cell deposited under American TypeCulture Collection Accession Number ATCC HB 11075 (referred to herein asD62.3.2).
 11. A composition according to claim 1, wherein the monoclonalantibody is present in a therapeutically effective amount.
 12. Acomposition according to claim 1, wherein the monoclonal antibody isbound to a cytotoxin.